Hydrazide-hydrazones have been described as a scaffold with antimicrobial and cytotoxic activities as well as iodinated compounds. A resistance rate of bacterial and fungal pathogens has increased considerably. That is why we synthesized and screened twenty-two iodinated hydrazide-hydrazones 1 and 2, ten 1,2-diacylhydrazines 3 and their three reduced analogues 4 for their antibacterial, antifungal, and cytotoxic properties. Hydrazide-hydrazones were prepared by condensation of 4-substituted benzohydrazides with 2-/4-hydroxy-3,5-diiodobenzaldehydes, diacylhydrazines from identical benzohydrazides and 3,5-diiodosalicylic acid via its chloride. These compounds were investigated in vitro against eight bacterial and eight fungal strains. The derivatives were found potent antibacterial agents against Gram-positive cocci including methicillin-resistant Staphylococcus aureus with the lowest values of minimum inhibitory concentrations (MIC) of 7.81 µM. Four compounds inhibited also human pathogenic fungi (MIC of ≥1.95 µM). The derivatives had different degrees of cytotoxicity for HepG2 and HK-2 cell lines (IC<sub>50</sub> values from 11.72 and 26.80 µM, respectively). Importantly, normal human cells exhibited lower sensitivity. The apoptotic effect was also investigated. In general, the presence of 3,5-diiodosalicylidene scaffold (compounds 1) is translated into enhanced both antimicrobial and cytotoxic properties whereas its 4-hydroxy isomers 2 share a low biological activity. N'-Benzoyl-2-hydroxy-3,5-diiodobenzohydrazides 3 have a non-homogeneous activity profile. Focusing on 4-substituted benzohydrazide part, the presence of an electron-withdrawing group (F, Cl, CF<sub>3</sub>, NO<sub>2</sub>) was found to be beneficial.